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Parameters affecting frequency of CRISPR/Cas9 mediated targeted mutagenesis in rice.
Frequency of CRISPR/Cas9-mediated targeted mutagenesis varies depending on Cas9 expression level and culture period of rice callus. Recent reports have demonstrated that the CRISPR/Cas9 system can function as a sequence-specific nuclease in various plant species. Induction of mutation in proliferating tissue during embryogenesis or in germline cells is a practical means of generating heritable mutations. In the case of plant species in which cultured cells are used for transformation, non-chimeric plants can be obtained when regeneration occurs from mutated cells. Since plantlets are regenerated from both mutated and non-mutated cells in a random manner, any increment in the proportion of mutated cells in Cas9- and guide RNA (gRNA)-expressing cells will help increase the number of plants containing heritable mutations. In this study, we examined factors affecting mutation frequency in rice calli. Following sequential transformation of rice calli with Cas9- and gRNA- expression constructs, the mutation frequency in independent Cas9 transgenic lines was analyzed. A positive correlation between Cas9 expression level and mutation frequency was found. This positive relationship was observed regardless of whether the transgene or an endogenous gene was used as the target for CRISPR/Cas9-mediated mutagenesis. Furthermore, we found that extending the culture period increased the proportion of mutated cells as well as the variety of mutations obtained. Because mutated and non-mutated cells might proliferate equally, these results suggest that a prolonged tissue culture period increases the chance of inducing de novo mutations in non-mutated cells. This fundamental knowledge will help improve systems for obtaining non-chimeric regenerated plants in many plant species.
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Genome-scale CRISPR-Cas9 screening in stem cells: theories, applications and challenges.
Due to the rapid development of stem cell technology, there have been tremendous advances in molecular biological and pathological research, cell therapy as well as organoid technologies over the past decades. Advances in genome editing technology, particularly the discovery of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-related protein 9 (Cas9), have further facilitated the rapid development of stem cell researches. The CRISPR-Cas9 technology now goes beyond creating single gene editing to enable the inhibition or activation of endogenous gene loci by fusing inhibitory (CRISPRi) or activating (CRISPRa) domains with deactivated Cas9 proteins (dCas9). These tools have been utilized in genome-scale CRISPRi/a screen to recognize hereditary modifiers that are synergistic or opposing to malady mutations in an orderly and fair manner, thereby identifying illness mechanisms and discovering novel restorative targets to accelerate medicinal discovery investigation. However, the application of this technique is still relatively rare in stem cell research. There are numerous specialized challenges in applying large-scale useful genomics approaches to differentiated stem cell populations. Here, we present the first comprehensive review on CRISPR-based functional genomics screening in the field of stem cells, as well as practical considerations implemented in a range of scenarios, and exploration of the insights of CRISPR-based screen into cell fates, disease mechanisms and cell treatments in stem cell models. This review will broadly benefit scientists, engineers and medical practitioners in the areas of stem cell research.
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Transcriptional gene fusions via targeted integration at safe harbors for high transgene expression in Chlamydomonas reinhardtii.
Conventional genetic engineering in green microalgae employs error-prone nonhomologous end joining to integrate recombinant DNA at double-strand breaks generated at random positions across the nuclear genome. This typically results in variable transcription strength and requires a labor-intensive screening procedure to identify transformants with sufficient expression. Current advances in genome editing enable scar-less integration of DNA at any desired locus for engineered bioproduction. We optimized construct design for predictable transgene expression at a high level, significantly improved scar-less integration rates into the nuclear genome via homology arm length optimization and quantified endogenous gene expression in vivo. Subsequently, endogenous genes were successfully targeted via Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9 (CRISPR/Cas9) to evaluate their capacity for high transgene expression and terpenoid production. Highest scar-less homology-directed repair efficiency was achieved with 50 bp homology arms. The Light harvesting Chl a/b binding protein of LHCII (LHCBM1) locus was found to be differentially expressed under several light intensities and allows an 8.6-fold increase in transgenic protein accumulation compared with random insertion approaches. Co-expression of a functional sesquiterpene synthase achieved a 60-fold increase in valencene production compared with previous attempts. We showed LHCBM1 locus constitutes a genetic safe harbor for transgene expression and demonstrates the potential of C. reinhardtii as a green cell factory.
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Bidirectional manipulation of gene expression in adipocytes using CRISPRa and siRNA.
OBJECTIVE: Functional investigation of novel gene/protein targets associated with adipocyte differentiation or function heavily relies on efficient and accessible tools to manipulate gene expression in adipocytes in vitro. Recent advances in gene-editing technologies such as CRISPR-Cas9 have not only eased gene editing but also greatly facilitated modulation of gene expression without altering the genome. Here, we aimed to develop and validate a competent in vitro adipocyte model of controllable functionality as well as multiplexed gene manipulation in adipocytes, using the CRISPRa "SAM" system and siRNAs to simultaneously overexpress and silence selected genes in the same cell populations. METHODS: We introduced a stable expression of dCas9-VP64 and MS2-P65, the core components of the CRIPSRa SAM system, in mesenchymal C3H/10T1/2 cells through viral delivery and used guide RNAs targeting Pparγ2, Prdm16, Zfp423, or Ucp1 to control the expression of key genes involved in adipocyte differentiation and function. We additionally co-transfected mature adipocytes with sgRNA plasmids and siRNA to simultaneously up-regulate and silence selected genes. Quantitative gene expression, oxygen consumption, fluorescence-activated cell sorting and immunocytochemistry served as validation proxies in pre- or mature adipocytes. RESULTS: CRISPRa SAM-mediated up-regulation of a key adipogenic gene, Pparγ2, was successfully achieved using selected sgRNAs targeting the Pparγ2 promoter region (i.e. up to 104 fold); this induction was long lasting and sufficient to promote adipogenesis. Furthermore, co-activation of Pparγ2 with either Prdm16 or Zfp423 transcripts drove distinct thermogenic gene expression patterns associated with increased or decreased oxygen consumption, respectively, mimicking typical characteristics of brite/beige or white cell lineages. Lastly, we demonstrated that up-regulation of endogenous genes in mature adipocytes was also easily and efficiently achieved using CRISPRa SAM, here exemplified by targeted Ucp1 overexpression (up to 4 × 103 fold), and that it was compatible with concomitant gene silencing using siRNA, allowing for bidirectional manipulation of gene expression in the same cell populations. CONCLUSIONS: We demonstrate that the CRISPRa SAM system can be easily adopted and used to efficiently manipulate gene expression in pre- and mature adipocytes in vitro. Moreover, we describe a novel methodological approach combining the activation of endogenous genes and siRNA-mediated gene silencing, thus providing a powerful tool to functionally decipher genetic factors controlling adipogenesis and adipocyte functions.
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Liganded Thyroid Hormone Receptors Transactivate the DNA Methyltransferase 3a Gene in Mouse Neuronal Cells.
Thyroid hormone (T3) is essential for proper neurological development. The hormone, bound to its receptors, regulates gene transcription in part by modulating posttranslational modifications of histones. Methylation of DNA, which is established by the de novo DNA methyltransferase (DNMT)3a and DNMT3b, and maintained by DNMT1 is another epigenetic modification influencing gene transcription. The expression of Dnmt3a, but not other Dnmt genes, increases in mouse brain in parallel with the postnatal rise in plasma [T3]. We found that treatment of the mouse neuroblastoma cell line Neuro2a[TRβ1] with T3 caused rapid induction of Dnmt3a mRNA, which was resistant to protein synthesis inhibition, supporting that it is a direct T3-response gene. Injection of T3 into postnatal day 6 mice increased Dnmt3a mRNA in the brain by 1 hour. Analysis of two chromatin immunoprecipitation-sequencing datasets, and targeted analyses using chromatin immunoprecipitation, transfection-reporter assays, and in vitro DNA binding identified 2 functional T3-response elements (TREs) at the mouse Dnmt3a locus located +30.3 and +49.3 kb from the transcription start site. Thyroid hormone receptors associated with both of these regions in mouse brain chromatin, but with only 1 (+30.3 kb) in Neuro2a[TRβ1] cells. Deletion of the +30.3-kb TRE using CRISPR/Cas9 genome editing eliminated or strongly reduced the Dnmt3a mRNA response to T3. Bioinformatics analysis showed that both TREs are highly conserved among eutherian mammals. Thyroid regulation of Dnmt3a may be an evolutionarily conserved mechanism for modulating global changes in DNA methylation during postnatal neurological development.
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CRISPR-mediated engineering of mesenchymal stromal/stem cells: a summary of recent progress in immunological applications for regenerative medicine and cancer therapy.
Mesenchymal stromal/stem cells (MSCs) have introduced as a cornerstone of regenerative medicine, owing to their immunomodulatory properties and therapeutic potential in autoimmune and inflammatory disorders. Although, their clinical application is often restricted due to immune rejection and heterogeneity in immunoregulatory responses. The advent of Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technology has revolutionized MSC engineering, enabling precise genetic modifications to enhance their immunological efficacy. This review explores how CRISPR-mediated editing of MSCs can mitigate immunogenicity, amplify anti-inflammatory functions, and repurpose MSCs for targeted immunotherapy. Key strategies include knockout of β2-microglobulin to evade T-cell recognition, augmentation of anti-inflammatory mediators like interleukin (IL)-10 and TNF-alpha stimulated gene/protein 6 (TSG-6), and disruption of pro-inflammatory pathways such as toll-like receptor 4 (TLR4)/NF-κB. In addition, CRISPR-engineered MSCs demonstrate promise in reshaping tumor microenvironments and combating bacterial infections through enhanced innate immunity. Despite challenges including off-target effects and delivery optimization, CRISPR-tailored MSCs represent a transformative approach to overcoming immunological barriers, paving the way for universal, off-the-shelf therapies in rheumatoid arthritis, cancer, and beyond.
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In Vivo Genome Editing Partially Restores Alpha1-Antitrypsin in a Murine Model of AAT Deficiency.
CRISPR (clustered regularly interspaced short palindromic repeats) genome editing holds promise in the treatment of genetic diseases that currently lack effective long-term therapies. Patients with alpha-1 antitrypsin (AAT) deficiency develop progressive lung disease due to the loss of AAT's antiprotease function and liver disease due to a toxic gain of function of the common mutant allele. However, it remains unknown whether CRISPR-mediated AAT correction in the liver, where AAT is primarily expressed, can correct either or both defects. Here we show that AAV delivery of CRISPR can effectively correct Z-AAT mutation in the liver of a transgenic mouse model. Specifically, we co-injected two AAVs: one expressing Cas9 and another encoding an AAT guide RNA and homology-directed repair template. In both neonatal and adult mice, this treatment partially restored M-AAT in the serum. Furthermore, deep sequencing confirmed both indel mutations and precise gene correction in the liver, permitting careful analysis of gene editing events in vivo. This study demonstrates a proof of concept for the application of CRISPR-Cas9 technology to correct AAT mutations in vivo and validates continued exploration of this approach for the treatment of patients with AAT deficiency.
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RNAS-sgRNA: Recurrent Neural Architecture Search for Detection of On-Target Effects in Single Guide RNA.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 is a leading genomic editing tool, but its effectiveness is limited by considerable heterogeneity in target efficiency among different single guide RNAs (sgRNA). This study presents RNAS-sgRNA, a hybrid model that integrates neural architecture search (NAS) with recurrent neural networks (RNN) to evaluate the on-target efficacy of CRISPR/Cas9 sgRNA. The RNAS-sgRNA model automates architectural discovery, improving sgRNA sequence categorization without considerable manual adjustment. The NAS component improves the RNN architecture, which analyzes sgRNA sequences represented as binary matrices and produces a classification score. Upon evaluation across several datasets, RNAS-sgRNA exhibits substantial performance enhancements with multiple cell lines, comparing its area under the receiver operating characteristic curve (AUROC) performance to the baseline CRISPRpred(SEQ) and DeepCRISPR models. RNAS-sgRNA demonstrated substantial improvements in AUROC performance in several cell lines compared with existing models. Notable improvements include enhancements of 8.62% for HCT116, 121.57% for HEK293T, 13.40% for HeLa, and 20.78% for HL60 cell lines, resulting in an overall improvement of 13.46%. Compared with DeepCRISPR, the model achieved additional AUROC gains in all cell lines tested, with an average improvement of 14.74%. The study also highlighted the ability of the model to deliver superior performance on smaller datasets through transfer learning, underscoring its potential applications in personalized medicine and genetic research. RNAS-sgRNA introduces a novel integration of NAS with RNN to evaluate the efficacy of CRISPR/Cas9 sgRNA. Unlike traditional methods that require significant manual adjustments, this model automates architectural discovery, optimizing the RNN structure for sgRNA sequence analysis. Furthermore, the application of transfer learning to fine-tune the pretrained model on small cell-line datasets represents a pioneering approach in the domain. The model's demonstrated ability to significantly outperform existing algorithms, including CRISPRpred(SEQ) and DeepCRISPR, across multiple cell lines highlights its innovative contribution to genomic editing research and personalized medicine.
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Generation of an Induced Pluripotent Stem Cell Line with the Constitutive EGFP Reporter.
The discovery of induced pluripotent stem cell (iPSC) technology has provided a versatile platform for basic science research and regenerative medicine. With the rise of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) systems and the ease at which they can be utilized for gene editing, creating genetically modified iPSCs has never been more advantageous for studying both organism development and potential clinical applications. However, to better understand the behavior and true therapeutic potential of iPSCs and iPSC-derived cells, a tool for labeling and monitoring these cells in vitro and in vivo is needed. Here, we describe a protocol that provides a straightforward method for introducing a stable, highly expressed fluorescent protein into iPSCs using the CRISPR/Cas9 system and a standardized donor vector. The approach involves the integration of the EGFP transgene into the transcriptionally active adeno-associated virus integration site 1 (AAVS1) locus through homology directed repair. The knockin of this transgene results in the generation of iPSC lines with constitutive expression of the EGFP protein that also persists in differentiated iPSCs. These EGFP-labeled iPSC lines are ideal for assessing iPSC differentiation in vitro and evaluating the distribution of iPSC-derived cells in vivo after transplantation into model animals.
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A Revolution toward Gene-Editing Technology and Its Application to Crop Improvement.
Genome editing is a relevant, versatile, and preferred tool for crop improvement, as well as for functional genomics. In this review, we summarize the advances in gene-editing techniques, such as zinc-finger nucleases (ZFNs), transcription activator-like (TAL) effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) associated with the Cas9 and Cpf1 proteins. These tools support great opportunities for the future development of plant science and rapid remodeling of crops. Furthermore, we discuss the brief history of each tool and provide their comparison and different applications. Among the various genome-editing tools, CRISPR has become the most popular; hence, it is discussed in the greatest detail. CRISPR has helped clarify the genomic structure and its role in plants: For example, the transcriptional control of Cas9 and Cpf1, genetic locus monitoring, the mechanism and control of promoter activity, and the alteration and detection of epigenetic behavior between single-nucleotide polymorphisms (SNPs) investigated based on genetic traits and related genome-wide studies. The present review describes how CRISPR/Cas9 systems can play a valuable role in the characterization of the genomic rearrangement and plant gene functions, as well as the improvement of the important traits of field crops with the greatest precision. In addition, the speed editing strategy of gene-family members was introduced to accelerate the applications of gene-editing systems to crop improvement. For this, the CRISPR technology has a valuable advantage that particularly holds the scientist's mind, as it allows genome editing in multiple biological systems.
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